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NIH, NICHD, LGRD, Bethesda, Maryland, USA
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The failure of sister chromosomal sites to separate in condensin mutants has traditionally been attributed to topological tangles between sister chromatids (Strunnikov 2003), inferred by analogy with the topoisomerase II mutant phenotype (Uemura et al. 1987). The occurrence of anaphase chromosomal bridges in condensin-deficient cells in Metazoa, however, is not as frequent as one would expect, taking into account the gross disarray of chromosome structure in prometaphase (Hagstrom et al. 2002; Hudson et al. 2003; Ono et al. 2003). Similarly, in budding yeast, while condensin mutants arrest at non-permissive conditions with partially separated sister chromatids (Freeman et al. 2000) (widely accepted to represent an anaphase delay), these cells do maintain high viability throughout the period of arrest (Freeman et al. 2000; Lavoie et al. 2002) (Fig. 1A). The nature of this pathway, which largely rescues segregation defects upon condensin dysfunction or depletion in eukaryotes, remains unknown.
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We undertook a detailed investigation of the mechanism mediating the cell-cycle arrest in condensin mutants. This analysis allowed us to reconcile the severity of condensation defects in S. cerevisiae condensin mutants (Freeman et al. 2000; Lavoie et al. 2000; Ouspenski et al. 2000; Bhalla et al. 2002) with their prolonged viability and high stability of non-rDNA chromosomes, upon return to permissive conditions (Freeman et al. 2000; Lavoie et al. 2002). It also elucidated the role of non-rDNA condensin sites in budding yeast. We showed that the spindle assembly checkpoint monitors the mitotic activity of condensin, thereby securing its function in genome segregation. Inactivation of spindle checkpoint genes has demonstrated that the essential condensin function in S. cerevisiae mitosis is not circumscribed to its role in rDNA segregation, but is required for the correct transmission of all chromosomes. The centromeric role of condensin is a part of the tension generator at the kinetochore and involves functional interaction with the Cse4 centromere histone. Thus, we have uncovered an interface between the condensin complex and kinetochore function essential for partitioning of the entire genome.
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Results |
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We confirmed that smc2-8 population arrests as two different cell types (Fig. 1A). The majority are mid-anaphase-like cells with partially separated sister chromatids and unsegregated nucleoli (Freeman et al. 2000) (Fig. 1A, top panel). These cells were shown to retain high viability (by elutriation and plating, data not shown). A smaller fraction is inviable cells (Strunnikov et al. 1995; Freeman et al. 2000), either unbudded or with a small bud (Fig. 1A, middle panel). The fraction of unbudded cells (and the corresponding cell viability) varies, dependent on the experimental conditions, but generally falls within 20%–50% range for this particular condensin allele. It has been suggested that these inviable cells are aneuploids (Strunnikov et al. 1995). Our analysis of nucleolar and/or chromosome XII segregation in smc2-8 supports this assessment, by showing that they either lack nucleoli or have very small nucleoli, probably a result of chromosome XII segregation errors and breaks (Fig. 1A, lower panel). Interestingly, a minor subpopulation composed of small-budded cells evidently initiated (but not finished) S-phase (Fig. 1A, right panel), a conclusion supported by budding, Pds1p presence and FACS profile of the arrested smc2-8 population (Fig. 1A). These cells, unlike the unbudded cells that lack visible nucleoli, have reduced-size nucleoli (Fig. 1A, right panel arrow). Diminution of nucleoli, accompanying S-phase block and loss of viability in these cells are likely related and probably can explain an indirect requirement of condensin (through its role in nucleolar maintenance) for robust DNA replication, which is not addressed in this work.
For the rest of the study we focused on the large-budded cells resulting from condensin inactivation, representing the fraction of the cell population arrested in mitosis. First, we tested the arrested condensin mutants for a biochemical marker of anaphase: cleavage and degradation of the cohesin subunit Mcd1p/Scc1p (Uhlmann et al. 1999; Rao et al. 2001). In nine independent condensin mutants the arrest was not consistent with uniform anaphase: the full-length Mcd1p levels remained high as compared to the cdc15 anaphase arrest (Fig. 1B), and the cleavage products were undetectable (not shown). Therefore, at least some condensin mutants must have a high proportion of metaphase cells.
Next, we undertook time course analysis of the synchronized smc2-8 cell population, in comparison to the wild-type. While wild-type cells released from G1 arrest proceeded synchronously through anaphase after 90 min (according to nuclear division kinetics, Pds1p and Mcd1p dynamics) and exited mitosis between 120 and 135 min (Clb2p degradation) (Fig. 1C,D), the condensin mutants did not indicate significant anaphase entry or exit from mitosis. To the contrary, mutant cells accumulated Pds1p, Mcd1p and Clb2p, consistent with prolonged metaphase arrest. This suggests that condensin mutants arrest in metaphase and that the partial sister chromatid separation in the arrested cells (Freeman et al. 2000; Bhalla et al. 2002) is largely a result of metaphase, not anaphase, spindle forces (Goshima & Yanagida 2000; He et al. 2000; Tanaka et al. 2000).
A metaphase arrest and high viability suggest that a certain checkpoint is activated in condensin mutants. As some specific mutant alleles of condensin subunits lead to DNA damage (Aono et al. 2002; Huang & Koshland 2003), we tested the possibility that condensin mutants arrest is a result of DNA damage checkpoint (Weinert et al. 1994). We constructed a triple smc2-8 chk1 rad53 mutant defective in condensin function, as well as in two branches of the DNA integrity checkpoint pathway (Sanchez et al. 1999; Early et al. 2004). The resulting strain displayed unaltered arrest morphology at 37 °C, did not form microcolonies at 37 °C (not shown), had viability at the same level as single mutants (Fig. 2A) and its growth recovery after the exposure to 37 °C was similar to the single smc2-8 mutants (Fig. 2B). The smc4-1 allele displayed similar properties (not shown). This result demonstrates that the mitotic arrest caused by these highly penetrant condensin alleles is not mediated by a DNA damage checkpoint.
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, resulting in synthetic lethality at 23 °C (Fig. 2F), suggesting that condensin mutants also require a mitotic exit delay for viability, even at the permissive temperature.
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smc2-8 cells exit the first mitosis (Fig. 3A), but undergo no more than two cell divisions after exposure to 37 °C (Fig. 3B). Moreover, comparison of the FACS profiles for the smc2-8 single and the bub1 smc2-8 double mutants (Fig. 3C) suggests substantial mis-segregation of individual chromosomes (indicated by the broadening peaks and appearance of the peak with less than G1 DNA content).
In order to compare the rates of mis-segregation for individual chromosomes we employed their in vivo labeling by RFP or GFP markers in the wild-type, bub1, smc2-8 and bub1 smc2-8 cells. For rDNA/Chr. XII segregation we simultaneously monitored nucleoli (Sik1p-mRFP) and spindle microtubules (Tub1p-GFP). The mis-segregation event was scored when the mitotic spindle appeared fully elongated but discontinuous, and the nucleolus was in only one of the dividing cells (Fig. 3D). For non-rDNA chromosomes IV and IX we combined DAPI staining with LacO/LacI-GFP tags. In this case the mis-segregation event was scored when the DAPI signal had separated to opposite polar positions in mother and daughter cells, but two LacI-GFP signals were detected in only one of the DAPI masses (Fig. 3E). While wild-type cells had no mis-segregation and bub1
displayed no more than 8% mis-segregation events, regardless of the chromosomal tag position, analysis of the smc2-8 mutant confirmed that the rDNA-containing chromosome XII was much more prone to mis-segregation than chromosomes IV or IX (Fig. 3F). In contrast, bub1 smc2-8 cells had equally high mis-segregation rates for chromosomes XII, IV and IX (Fig. 3F).
Thus, condensin in budding yeast is essential for segregation of the whole genome (not just the rDNA chromosome), accounting for its presence on every S. cerevisiae chromosome (Wang et al. 2005). As it is well-established that the spindle checkpoint signal is generated at the kinetochore (Tan et al. 2005), it is conceivable that condensin is required for some aspect of kinetochore function. As condensin is a chromatin protein, it can potentially affect kinetochores through its cis-activity at CEN-neighboring sequences. Therefore, condensin mutations may lead to altered pericentromeric chromatin, resulting in spindle defects due to disruption of spindle-kinetochore attachment and/or metaphase tension. Such defects are indirectly suggested by experimental data: smc2-8 and smc4-1 alleles display notable sensitivity to low doses of a microtubule polymerization inhibitor (Fig. 4A), a sign of diminished spindle stability. Thus, we employed a more sensitive genetic assay to assess the spindle-kinetochore tension defects in condensin mutants: interaction with the sgo1 (Kitajima et al. 2004; Indjeian et al. 2005) and skp1-AA alleles (Kitagawa et al. 2003), which are unable to transmit a signal of inadequate tension. Double sgo1 smc2-8 and skp1-AA smc2-8 mutants were indeed unable to recover from the 37 °C shift, due to a progressive and rapid decrease in cell viability (Fig. 4B,C). These double mutants also bypassed the metaphase arrest, as assayed by nuclear division and Western-blotting (not shown). Further analysis was performed for the skp1-AA mutant, as this allele, apparently, confers a very targeted disruption of the tension sensory mechanism, with no effect on the essential kinetochore functions of Skp1p (Kitagawa et al. 2003). Using a colony color-sectoring assay, we observed a significant increase in the loss rate of a non-essential chromosome fragment in the double mutant in comparison to the wild-type or single mutants (Fig. 4D).
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The nature of the spindle-kinetochore tension defect in condensin mutants is probably related to the centromere structure, as condensin is a chromatin component. In the absence of comprehensive morphological tests for centromere and kinetochore structure in S. cerevisiae it is difficult to identify the underlying molecular defect, unless there is a substantial loss of kinetochore proteins. Our Slk19p-GFP analysis (Fig. 4E) did not suggest such a significant defect. It is also evident that disruption of the kinetochore structure in smc2-8 is unlikely to be dramatic, because some cells do go through anaphase (Fig. 1) and there is no indication of gross chromosome loss in the single mutant (Freeman et al. 2000). However, as kinetochore structure is extremely complex, it possible that some factors required for kinetochore assembly and bipolar attachment are not equally required for maintenance (during metaphase arrest), and therefore their loss can be detected. Thus, we monitored the localization of GFP fusions (Huh et al. 2003) of other representative subunits of kinetochore subcomplexes (Kline-Smith et al. 2005) in smc2-8. As condensin-deficient yeast cells do maintain centromere clustering (data not shown and Figs 4E and 5A), it was feasible to visualize these low-abundance proteins as fluorescent dots in the nuclei of metaphase-arrested cells. After 3-h exposure of smc2-8 cells to 37 °C no dramatic localization changes were observed for Dad1p (DDD complex) (Fig. A), Ame1p (COMA complex), Nuf2p, Spc24p, Spc25p (all Ndc80 complex) or inner kinetochore proteins (Ndc10p, Cbf2p, Ctf3p and Mif2p), yet some un-clustering of kinetochores was observed in a fraction of cells (data similar to Fig. 5A, not shown). Limited dissipation of the GFP signal was also observed for MIND complex components Dsn1p and Mtw1p (confirmed by ChIP, not shown).
In contrast, localization of Cse4p, the centromere histone, was significantly altered in smc2-8 at 37 °C, so that Cse4p-GFP, in addition to a weakened signal at the centromere cluster, displayed a diffuse signal throughout the nucleus (Fig. 5B). No such delocalization of Cse4p was observed in the wild-type, even upon metaphase delay due to expression of the dominant non-cleavable cohesin subunit Mcd1p (Fig. 5B, upper panel), ruling out the possibility that Cse4p delocalization in smc2-8 is an indirect consequence of the prolonged mitotic arrest itself. The diffuse nuclear distribution of Cse4p-GFP is attributable to either its over-expression (Collins et al. 2004, 2007) or its loss from centromeric chromatin (Crotti & Basrai 2004). The former was ruled out by showing that the amount of full-length Cse4p-GFP is unchanged in smc2-8 cells compared to wild-type (Fig. 5C). Conversely, partial Cse4p-GFP depletion from centromeric chromatin in smc2-8 was readily demonstrated by ChIP analysis (Fig. 5E). The qPCR/ChIP data show that centromere chromatin (at CEN4) at 37 °C is 50% less enriched for Cse4p-GFP in smc2-8 than in wild-type cells. The result was even more pronounced for CEN12 (Fig. 5F). Such a significant Cse4p depletion from the centromere (Fig. 5E,F) would generally exert a detrimental effect on the kinetochore assembly (Pinsky et al. 2003; Crotti & Basrai 2004; Collins et al. 2005), but has never been demonstrated or characterized in metaphase (fully assembled) centromere. General localization stability of GFP-fused kinetochore proteins in our analysis suggests that once sister kinetochores are already assembled and properly attached to the spindle (in smc2 metaphase arrest), most kinetochore proteins are able to sustain the observed twofold reduction in Cse4p levels. Moreover, it is likely that the observed loss of Cse4 is limited to exchangeable nucleosomes, which are not in the centromere core (Fig. 6).
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Discussion |
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Condensin activity in vivo has been shown to be essential for chromosome compaction and segregation (Sutani et al. 1999; Freeman et al. 2000; Lavoie et al. 2000; Ouspenski et al. 2000; Hagstrom et al. 2002; Coelho et al. 2003; Hudson et al. 2003; Ono et al. 2003; Savvidou et al. 2005). In all of these studies the disruption of condensin function manifested in cytological defects in the prometaphase and anaphase: abnormal chromosome condensation and anaphase bridging, respectively. Importantly, some of the studies in higher eukaryotes have demonstrated that condensin-depleted cells exhibit a notable mitotic delay (Hudson et al. 2003; Ono et al. 2003; Hirano 2005), the mechanism of which was unknown. It was proposed that condensin defects in these systems generate merotelic attachment (Salmon et al. 2005) of microtubules to the kinetochore, which, despite being aberrant, nevertheless does not activate the spindle checkpoint (Hirano 2005). Our preliminary results show, however, that condensin deficiency does trigger a spindle checkpoint-dependent mitotic delay in cultured human cells (Ilia Ouspenski, unpublished). On the other hand, since the discovery of the first chromosomal condensin binding site, the rDNA locus in budding yeast, it was perplexing to see the apparent bias in condensin function towards rDNA segregation (Freeman et al. 2000). The present study apparently resolves both of these issues: the nature of mitotic delay/arrest and the bias in rDNA mis-segregation upon condensin dysfunction.
The demonstration that mutations in condensin, when combined with mutations in spindle checkpoint genes, bring about bypass of metaphase arrest, chromosome loss and lethality, proves that condensin function is monitored by a mitotic surveillance mechanism. Most notably, we found strong genetic interaction of the smc2-8 allele with the bub1, mad1, mad2 and mad3 null mutations, defective in monitoring bipolar orientation of chromosomes (Shonn et al. 2003; Gillett et al. 2004; Howell et al. 2004). The unveiled genetic interactions of smc2-8 with skp1-AA or sgo1 alleles, which impair the detection of tension defects (Kitagawa et al. 2003; Indjeian et al. 2005), and the increased separation of sister kinetochores in metaphase in smc2-8 (Fig. 4), both suggest that condensin is required for proper tension at the kinetochore. At the same time, the high viability of condensin mutants indicates that spindle-kinetochore attachment and sister kinetochore orientation are maintained at nearly wild-type fidelity in checkpoint-proficient condensin mutants.
The synthetic lethality between the smc2-8 allele and the BUB2 pathway could suggest that condensin activity is also monitored at the late stages of mitosis. The Bub2 protein, in complex with Bfa1p, is a regulator of the mitotic exit network (MEN) (Wang et al. 2000; Pereira et al. 2002). As condensin was shown to be required not only for establishment but also for maintenance of condensation in metaphase (Freeman et al. 2000) and anaphase (Machin et al. 2005), it is possible that the BUB2 pathway is required to monitor condensin function in chromosomal regions that segregate late in anaphase, such as telomeres and the nucleolus. Further investigation is required to elucidate the mechanism of interaction between the condensin pathway and the BUB2 checkpoint.
The results of the present study offer a plausible explanation for the noticeable mis-segregation of an rDNA chromosome in checkpoint-proficient condensin mutants, while non-rDNA chromosomes maintain a high fidelity of segregation (Freeman et al. 2000). As the ultimate function of a checkpoint is to provide an unscheduled or extended cell cycle delay to repair specific damage (Elledge 1996), we can hypothesize that relatively mild condensin-mediated defect at the centromeres (i.e. altered tension) triggers such a delay. Our work suggests that some condensin function (probably at the sites proximal to natural centromeres) is required for proper kinetochore structure and thus such sites could serve as "circuit breakers" engaging spindle checkpoint upon condensin failure (Fig. 6). The mitotic delay generated this way should allow correction of the negative effect of condensin mutations on chromosome arm segregation. However, the nucleolar segregation block is not efficiently repaired during such a delay, probably due to constant regeneration of segregation impediment as a result of active rDNA transcription (Tomson et al. 2006; Wang et al. 2006). Such ineffectiveness of a metaphase delay for repair of condensin dysfunction in the transcriptionally active nucleolus in S. cerevisiae may be an evolutionary inducement for developing a mitotic nucleoli-reorganization pathway in multicellular organisms (Dimario 2004).
Condensin activity is required for robust centromere function
In several higher systems condensin either co-localizes with the centromere (Ono et al. 2004) or facilitates proper chromosome arrangement on the spindle (Hagstrom et al. 2002; Wignall et al. 2003; Ono et al. 2004). However, in all these systems centromeres are immense structures and the specificity of condensin defects for centromeric chromatin vs. overall chromosomal defects, is difficult to isolate. Our recent findings that S. cerevisiae condensin is enriched at centromeres in general, localizes in close proximity to particular centromeres and that pericentromeric sites display a mitotic increase in condensin binding (Wang et al. 2005) suggested that condensin activity can be an important supplement to mitotic centromere functionality. The present work supports this hypothesis by showing that the spindle checkpoint is activated in condensin mutants, these mutants have a kinetochore tension defect, chromosome non-disjunction in condensin/checkpoint double mutants is chromosome-size-independent and centromere proteins are lost in condensin mutation-mediated arrest. Data showing that the mitotic spindle checkpoint is also activated by condensin depletion in human cells (Ilia Ouspenski, unpublished) indicates that the role for condensin in centromere maintenance and proper kinetochore tension may be conserved in evolution.
The exact molecular function of the centromere-proximal condensin binding sites in wild-type yeast chromosomes remains to be uncovered. However, it is unlikely that in S. cerevisiae condensin is an essential centromere component per se, as damage to kinetochore appears to be quite limited (Fig. 5A,D) in the smc2-8 mutant and there are no obvious spindle-attachment defects. Nevertheless, the interface between the condensin activity and the kinetochore appears to be in centromeric chromatin, that is, chromatin enriched in Cse4p, a CENP-A homologue. While the exact stoichiometry of Cse4p per each centromere is still controversial, the recent study (Riedel et al. 2006) shows that, unlike commonly assumed (Meluh et al. 1998; Joglekar et al. 2006) based largely on the small size of centromere footprint and low-resolution ChIP data, Cse4p is not limited to one nucleosome, but instead can occupy a more extensive region with a prominent peak centered at the CEN DNA, probably corresponding to the positioned Cse4 nucleosome at the centromere core. Our ChIP data show quite a narrow window of Cse4p enrichment at the CEN4 (Fig. 5E), but much broader at CEN12 (Fig. 5F), in both cases being consistent with rather extended Cse4p-bound region (i.e. several nucleosomes). Therefore, based on the most comprehensive analysis of Cse4p occupancy to date (Riedel et al. 2006) (and partially on our ChIP data) we can assume, that in addition to its core centromeric position, Cse4p is also incorporated in the broader peri-centromeric region, commonly referred to as centromeric chromatin in other species. This places Cse4p-enriched chromatin in close proximity to the pericentromeric condensin binding peaks (Wang et al. 2005). Therefore, it is plausible that condensin activity affects kinetochore tension not through a direct condensin-kinetochore protein contact, but is mediated by centrometric chromatin. In support of this hypothesis, a recent Drosophila study shows the possibility of direct interaction between the CAP-G condensin subunit and CID (Cse4p ortholog) (Jager et al. 2005), while no reliable interaction of condensin with kinetochore proteins has been reported. Condensin's involvement in the generation of proper tension at native kinetochores could be in establishing localized condensation of Cse4p-enriched chromatin (Fig. 6A).
Another interesting phenomenon discovered in the course of our study is that complete saturation of centrometric chromatin by Cse4p may not be necessary for the maintenance of the spindle-kinetochore attachment and orientation once they are established. It is known that Cse4p is required for formation of functional kinetochore (Collins et al. 2005); however, the smc2 mutant apparently has robust maintenance of proper orientation of sister kinetochores (as well as stable localization of kinetochore proteins), while up to 50% of Cse4p is lost from the centromeres. This result could be related to the fact that only a small subpopulation of Cse4p (and CENP-A) has direct interaction with kinetochore proteins (Fig. 6). This is suggested by the results of several yeast proteomic screens that failed to uncover robust physical interactions between kinetochore proteins and Cse4p, as well as by the recent comprehensive analysis of CENP-A interactions in mammalian cells, which shows no biochemical evidence of complexes with known kinetochore components (Foltz et al. 2006). The model in Fig. 6B adequately explains the induction of spindle checkpoint, extended sister kinetochore separation while more distant regions of sister chromatids remain associated by cohesion, partial loss of Cse4p from centromeres and stability of kinetochore structure in condensin mutants. It could also give a direction to future analysis of the interface between Cse4p-containing chromatin and pericentromeric condensin binding (Wang et al. 2005), as well as between Cse4p and the kinetochore itself.
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Experimental procedures |
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All the strains used are isogenic to S288c unless otherwise indicated (Table 1). To analyze the genetic interaction between condensin mutations and the spindle checkpoint pathway, the deletions in BUB1, BUB2, MAD1 and MAD3 genes were first complemented with the respective wild-types genes on a CEN plasmid. The null mutants strains were then transformed with the linearized vector pLF733 (Wang et al. 2005) replacing the wild-type SMC2 gene with the smc2-8::LEU2. The minichromosomes bearing the wild-type checkpoint genes were then counter-selected. A similar approach was used to introduce the bub1 deletion into the ycs4-2 and brn1-9 mutants, except the multi-copy plasmids pAS1024 and pAS1062, expressing YCS4 and BRN1 respectively, were introduced before the BUB1 gene was disrupted as described (Brachmann et al. 1998). Similarly, the smc2-8 mutation (pLF733) was introduced into bub2 background after complementing this deletion in BY4741 with a BUB2 plasmid (pMA1183, A. Hoyt). The combined mutants rad53- chk1- smc2-8, skp1-AA smc2-8 and sgo1- smc2-8 were obtained through genetic crosses. sgo1 handling was according to Kitajima et al. (2004). Chromosome III fragment loss assay was performed as described by Freeman et al. (2000), after a 3 h incubation at 37 °C. For time-course cell cycle analysis cells were pre-synchronized by treating Bar1– or Bar1+ strains with 
factor (50 ng/mL or 250 ng/mL, respectively) for 2 h at 23 °C, followed by 1 h at 37 °C.
Biochemical methods
Protein analysis of crude cell extracts (prepared in 2% SDS) or immunoprecipitates was performed by Western blotting. Anti-Mcd1p antibody was from Guacci et al. (1997), anti-Clb2p and Cdc28p were from Santa Cruz Biotechnology (Santa Cruz, CA). Cse4p-GFP immunoprecipitation was performed according to an established method (Kagansky et al. 2004) with polyclonal anti-GFP antibodies (BD Biosciences). The qPCR/ChIP procedure was performed as described (Wang et al. 2004), with extracts from smc2-8 cells expressing Cse4p-GFP or Dad1p-GFP that were incubated at 37 °C for 3 h after synchronous release from G1 arrest. The ChIP controls included isogenic untagged strains, as well as the Smc2+ strain with the pAK727 plasmid expressing non-cleavable MCD1-9 allele (deletion of codons 177–271) under the MET15 promoter control. Ectopic induction of the MCD1-9 for 3 h results in prolonged mitotic arrest, unlike the SCC1-RRDD mutant described in Uhlmann et al. (1999), which exits mitosis after a transient delay (O. Cohen-Fix, personal communications). All controls were also exposed to 37 °C for 3 h.
Microscopy
The fluorescent markers for the nucleolus (Sik1p-mRFP) (Huh et al. 2003), mitotic spindle (Tub1p-GFP) and the LacI-GFP/LacO tags (Straight et al. 1996) were described previously. Pds1p-GFP was introduced using the pAC256 construct (Quimby et al. 2005). To evaluate segregation of different chromosomal loci (Fig. 3), cells were fixed and stained with DAPI as in Indjeian et al. (2005). The fluorescence was visualized using a Zeiss AxioVert epifluorescence microscope with a cooled CCD camera and OPENLAB image analysis software. Twenty Z-axis stacked images of each sample were taken. At least 100 cells were analyzed to quantify the nucleolar and chromosomal morphology. The GFP screen for kinetochore protein localization was performed in a set of strains that express different kinetochore proteins tagged with the GFP (Huh et al. 2003). Cultures of 15-mL were incubated at 37 °C for 3 h and then analyzed by fluorescent microscopy. For the subsequent analysis of Cse4p and Dad1p fusions, cells were synchronously released from -factor arrest at 37 °C and monitored for GFP localization every 30 min. The control Smc2+ strain was subjected to the same protocol, except it expressed non-cleavable MCD1-9 allele (pAK727 plasmid).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: strunnik{at}mail.nih.gov
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References |
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